Kits for measuring cellular transmembrane potential changes

ABSTRACT

The invention encompasses kits for an improved method for measuring membrane potential using compounds of the formula I as potentiometric probes. These probes may be used in combination with other fluorescent indicators such as Indo-1, Fura-2, and Fluo-3, such probes may be used in microplate reading devices such as FLIPR™, fluorescent imaging plate reader, sold by Molecular Devices Corp., of Sunnyvale, Calif.; flow cytometers; and fluorometers. Such probes are used to measure membrane potential in live cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. patent application Ser. No.09/924,797, filed Aug. 8, 2001, which is hereby incorporated byreference.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates generally to the fields of biology andchemistry, and bioanalytical instrumentation. In particular, the presentinvention is directed to composition and methods for use in sensingmembrane potentials, especially in biological systems. Potentiometricoptical probes enable researchers to perform membrane potentialmeasurements in organelles and in cells that are too small to allow theuse of microelectrodes. Moreover, in conjunction with imagingtechniques, these probes can be employed to map variations in membranepotential across excitable cells and perfused organs with spatialresolution and sampling frequency that are difficult to achieve usingmicroelectrodes.

2. Background of the Art

The plasma membrane of a cell typically has a transmembrane potential ofapproximately −70 mV (negative inside) as a consequence of K⁺, Na⁺ andCl⁻ concentration gradients that are maintained by active transportprocesses. Potentiometric probes offer an indirect method of detectingthe translocation of these ions. Increases and decreases in membranepotential (referred to as membrane hyperpolarization and depolarization,respectively) play a central role in many physiological processes,including nerve-impulse propagation, muscle contraction, cell signalingand ion-channel gating (references 1-3). Potentiometric probes areimportant tools for studying these processes, and for cell-viabilityassessment. Potentiometric probes include the cationic or zwitterionicstyryl dyes, the cationic carbocyanines and rhodamines, the anionicoxonols and hybrid oxonols and merocyanine 540 (references 4-8). Theclass of dye determines factors such as accumulation in cells, responsemechanism and toxicity. Mechanisms for optical sensing of membranepotential have traditionally been divided into two classes: sensitivebut slow redistribution of permanent ions from extracellular medium intothe cell, and fast but small perturbation of relatively impermeable dyesattached to one face of the plasma membrane (references 2 and 3).

The bis-barbituric acid and thiobarbituric oxonols, often referred to asDiBAC and DiSBAC dyes respectively, form a family of spectrally distinctpotentiometric probes with excitation maxima covering most of the rangeof visible wavelengths. DiBAC₄(3) and DiSBAC₂(3) have been the two mostpopular oxonol dyes for membrane potential measurement (references 9 and11). These dyes enter depolarized cells where they bind to intracellularproteins or membranes and exhibit enhanced fluorescence and red spectralshifts. Increased depolarization results in more influx of the anionicdye and thus an increase in fluorescence. DiBAC₄(3) reportedly has thehighest voltage sensitivity. The long-wavelength DiSBAC₂(3) hasfrequently been used in combination with the UV light-excitable Ca²⁺indicators Indo-1 or Fura-2 for simultaneous measurements of membranepotential and Ca²⁺ concentrations. Interactions between anionic oxonolsand the cationic K⁺-valinomycin complex complicate the use of thisionophore to calibrate potentiometric responses. DiBAC and DiSBAC dyesare excluded from mitochondria because of their overall negative charge,making them superior to carbocyanines for measuring plasma membranepotentials.

In general, DiBAC and DiSBAC dyes bearing longer alkyl chains had beenproposed to have better properties for measuring membrane potentials(references 5 and 12). DiSBAC₆(3) has been selected to use in aFRET-based membrane potential assay (reference 12). There are no reportson DiBAC₁ and DiSBAC₁ for measuring membrane potential.

It has been discovered that DiBAC₁(3) and DiSBAC₁(3) that possessunexpected properties that can be used to measure membrane potentialswith FLIPR and other fluorescence devices. Compared with other membersof the DiBAC and DiSBAC family, DiBAC₁ (3) and DiSBAC₁(3) give strongersignal and faster response, and exhibit greater water solubility.

SUMMARY

The invention encompasses an improved method for measuring membranepotential using compounds of formula I as potentiometric probes. Theseprobes may be used in combination with other fluorescent indicators suchas Indo-1, Fura-2, and Fluo-3, CALCIUM GREEN or Fluo-4. Such probes maybe used in microplate reading devices such as the FLIPR fluorescentimaging plate reader, sold by Molecular Devices Corp. of Sunnyvale,Calif.; flow cytometers; and fluorometers. Such probes are used tomeasure membrane potential in live cells.

The invention also encompasses test kits containing reagents of formulaI, and reagents of formula I in combination with another fluorescentreagent, in particular where the fluorescent reagent is a fluorescentindicator such as Indo-1, Fura-2, Fluo-3, CALCIUM GREEN or Fluo-4.

Another aspect of the invention involves a method for generatingvoltage-sensitive fluorescent changes comprising incubating the membranewith:

-   -   (a) A first reagent selected from the potentiometric probes        which redistribute from one side of the membrane to the opposite        side in response to transmembrane potential; and a second        reagent selected from the group consisting of non-fluorescent        dyes or pigments that are not membrane-permeable, and undergo        energy transfer with the first reagent on one side of the        membrane to reduce or eliminate the fluorescence signal on that        side; or    -   (b) A first reagent selected from the potentiometric probes        which redistribute from one side of the membrane to the opposite        side in response to transmembrane potential; and a second        reagent selected from the group consisting of non-fluorescent        dyes or pigments that are not membrane-permeable, and that        absorb the excitation light or emission from the first reagent        on one side of the membrane to reduce or eliminate the undesired        fluorescence signal; or    -   (c) A first reagent selected from the potentiometric probes        which redistribute from one side of the membrane to the opposite        side in response to transmembrane potential; and a second        reagent selected from the group consisting of fluorescent or        luminescent probes which undergo energy transfer with the first        reagent, said second reagent being located adjacent to either        the one side or the other side of the membrane.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a reaction scheme for making DiBAC and DiSBAC.

FIG. 2 illustrates the voltage-dependent fluorescence intensity changesof DiSBAC₁(3) in P2X2 cells at different ATP concentrations.

FIG. 3 illustrates the absorption spectra of DiSBAC₁(3), DiSBAC₂(3),DiSBAC₄(e), DiBAC₁(3) and DiBAC₄(3) in 1:1 methanol/water.

FIG. 4 illustrates the fluorescence spectra of DiSBAC₁(3), DiSBAC₂(3),DiSBAC₃(3), DiBAC₄(3) DiBAC₁(3) and DiBAC₄(3) in 1:1 methanol/water.

DETAILED DESCRIPTION

Compounds useful in practicing the present invention are made accordingto methods described by G. W. Fischer in Chem Ber, 1969, 102: 2609-2620,as shown in FIG. 1.

The invention is illustrated by the following examples:

EXAMPLE 1 Preparation of DiSBAC₁(3)

DiBAC and DiSBAC dyes are prepared based on the procedure for ethyl andbutyl derivatives (H. Bartsch and G. Haubold, Arch. Pharm. 1982, 315,761-766). Specifically, malonaldehyde bis(phenylimine)monohydrochloride(2.6 g, 10 mmol) and 1,3-dimethyl-2-thiobarbituric acid (3.5 g, 20 mmol)are dissolved in acetonitrile (40 mL). To the solution is addedtriethylamine (2 g, 20 mmol). The reaction mixture is refluxed until thestarting materials are completely consumed as indicated by TLC. Themixture is cooled to room temperature, and poured into acidic water (pH2-3, 350 mL). The resulting suspension is filtered to collect the solidthat is washed with cold water and air-dried. The crude product isfurther purified on a silica gel column using a gradient ofdichloromethanol/methanol to give the desired product.

DiSBAC₁(5), DiBAC₁(3), DiBAC₁(5) and other oxonol dyes are preparedanalogous to the above procedure.

EXAMPLE 2 Measuring Membrane Potentials using DiSBAC₁(3) in Combinationwith the Fluorescence Imaging Plate Reader (FLIPR™)

This specific example illustrates how to use DiSBA₁(3) in P2X2 cells incombination with the FLIPR™ fluorescent imaging plate reader sold byMolecular Devices Corp. of Sunnyvale, Calif. P2X2 cells are 1321 Niastrocytoma cells transfected to overexpress the purinergic P2X2ligand-gated ion channel. P2X2 belongs to a class of purinergic ionchannels that pass calcium and sodium in response to purine, includingadenosine 5′-triphosphate (ATP). P2X2 cells are propagated andmaintained in DME (high glucose), 10% FCS, 1× Pen/Strep and 2 mML-glutamine. Doubling time is approximately 36 hours. P2X2 cells shouldbe split at a 1 to 2 ratio upon confluence. The cells should be carriedfor no more than 20 passages. When approaching this limit, a new frozenvial of cells should be resurrected. Following is a typical kitprocedure:

-   -   1. Plate 40,000 P2X2 cells in 100 μL per well for 96 well plates        or 10,000 P2X2 cells in 25 μL per well for 384 well plates for        overnight.

2. Prepare 1× Loading Buffer.

-   -   2.1 To prepare the 1× Assay Buffer, pipette 10 mL of 10× Reagent        Buffer (1× Hanks' Balance Saline Solution+20 mM HPEPES, pH 7.40)        and dilute in 90 mL of distilled water. Adjust pH to 7.4 using        1.0 N NaOH and/or 1.0 N HCl.    -   2.2 To prepare 10 mM stock solution of DiSBAC₁(3), dissolve 3.8        mg DiSBAC₁(3) in 1 mL DMSO    -   2.3 To prepare 10% pluronic acid, dissolve 400 mg pluronic acid        in 4 mL water. Heat to 37° C. to complete dissolution.    -   2.4 To prepare 1× Loading Buffer, add 30 μL of stock DiSBAC₁(3),        8 μL 10% pluronic acid, and 20.0 mg DB71 in the 1× FLIPR assay        buffer.    -   3. Load Cells with 1× Loading Buffer    -   4. Remove cell plates from the incubator    -   5. Add 100 μL of 1× Loading Buffer per well of 96-well plates or        25 μL per well of 384-well plates.    -   6. Incubate plates at 37° C. for 30 minutes.    -   7. Run the FLIPR Membrane Potential Assay    -   7.1 Make 5× compound plate prior to running the FLIPR assay.        Dissolve 27.5 mg of ATP (Sigma Cat# A3377) in 1 mL of sterile        water to make a 50 mM stock solution. Make appropriate dilutions        for 100 nM, 1 μM and 10 μM and transfer a minimum of 200 μL to        each well of a compound plate.    -   7.2 Confirm that Membrane Potential Filter is installed. Choose        p2x2.fcf for experimental setup in the FLIPR software. Set up        the appropriate experiment parameters.    -   7.3 After incubation, transfer the plates directly to FLIPR and        begin the Membrane Potential Assay.    -   8. Run FLIPR and perform data analysis.

The curve of the ATP dose response should look similar to that shown inFIG. 2; an initial depolarization event depicted as an increase influorescence followed by repolarization or decay in signal nearbaseline. Also, EC50 should be approximately in the 10-100 nM range.

EXAMPLE 3 Measuring Membrane Potentials Using DiBAC₁(3) in Combinationwith a Microscope

DiBAC₁(3) is used to measure membrane potential change with a microscopeas described by L. M. Loew (Methods in Cell Biology, vol. 38, pp195-209).

EXAMPLE 4 Measuring Membrane Potentials Using DiSBAC₁(3) in Combinationwith Flow Cytometer

DiBAC₁(3) is used to measure membrane potential change with a flowcytometer as described by L. M. Loew (Methods in Cell Biology, Vol. 41,Part A, pp 195-209).

EXAMPLE 5 Water Solubility and Hydrophobicity Comparison of DiBAC andDiSBAC Dyes

DiSBAC₁(3), DiSBAC₂(3), DiSBAC₃(3), DiSBAC₄(3), DiBAC₁(3) and DiBAC₄(3)are dissolved in DMSO (3 mM). The DMSO stock solutions are respectivelypartitioned in 1:1 octanol/water mixture. The concentrations of theoxonol dyes in octanol and water layers are determined by absorptionspectra. The results are summarized in the following table I. As shownin the table, DiSBAC₁(3) and DiSBAC₁(3) are much more hydrophilic thanthe other oxonol dyes. They also have much better water solubility.TABLE 1 λ-max Absorbance in Water/ octanol Absorbance Absorbance inAbsorbance in Relative Compound (nm): in Water: Octanol: Octanol Values:DiBAC₁(3) 495 1.297 0.3104 4.179 1 DiBAC₄(3) 497 0.002501 2.854 0.000880.00021 DiSBAC₁(3) 538 0.06147 0.9126 0.0674 1 DiSBAC₂(3) 543 0.011432.187 0.0052 0.078 DiSBAC₃(3) 544 0.002256 4.331 0.0005 0.0077DiSBAC₄(3) 544 0.003318 3.226 <0.0010 <0.0077

EXAMPLE 6 Absorption Comparison of DiBAC and DiSBAC Dyes

DiSBAC₁(3), DiSBAC₂(3), DiSBAC₃(3), DiSBAC₄(3), DiBAC₁(3) and DiBAC₄(3)are dissolved in methanol (1 mM). The stock solutions are diluted with1:1 methanol/water, and the absorption spectra are recorded in aspectrophotometer. As shown in FIG. 3, DiBAC₁(3) and DiSBAC₁(3) possessunexpected blue shift compared to the other oxonol dyes.

EXAMPLE 7 Fluorescence Comparison of DiBAC and DiSBAC Dyes

DiSBAC₁(3), DiSBAC₂(3), DiSBAC₃(3), DiSBAC₄(3), DiBAC₁(3) and DiBAC₄(3)are dissolved in methanol (1 mM). The stock solutions are diluted with1:1 methanol/water, and the absorption spectra are recorded in afluorometer. As shown in FIG. 4, DiBAC₁(3) and DiSBAC₁(3) possessunexpected blue shift compared to the other oxonol dyes.

EXAMPLE 8 Fluorescence Response Comparison of DiBAC and DiSBAC dyes forSensing Membrane Potentials in FLIPR Assays

DiSBAC₁(3), DiSBAC₂(3), DiSBAC₃(3), DiSBAC₄(3), DiBAC₁(3) and DiBAC₄(3)are dissolved in DMSO (1 mM). The stock solutions are respectively usedto assay membrane potential changes in the P2X2 cells in combinationwith FLIPR™ as described in Example 2. The results are summarized in thefollowing table 2. TABLE 2 Fluorescence enhancement by 10 Compounds μMATP stimulation (in folds) Response speed DiBAC₁(3) 3.7 fast DiBAC₄(3)3.3 slow DiSBAC₁(3) 108.5 fast DiSBAC₂(3) 38.6 moderate DiSBAC₃(3) 14.5slow DiSBAC₄(3) 2.3 slow

As shown in the Table 2, DiSBAC₁(3) is much more sensitive, and hasfaster response to membrane potential change than the rest of theDiSBACs. DiBAC₁(3) also has much faster response to membrane potentialchange than DiBAC₄(3).

EXAMPLE 9 Use of DiSBAC₁(3) as a Fluorescent Indicator of TransmembranePotential Depolarization of PC 12 Cells

Protocols for transmembrane potential measurements are summarizedbriefly since they are similar to those given in detail in Example 1above. The Bis-(1,3-dimethylthiobarbituric acid)trimethine oxonol,(DiSBAC₁(3)), fluorescent reagent may be purchased from Molecular Probes(Eugene, Oreg., USA). The 1× Cell-Loading Buffer for DiSBAC₂(3),consists of sodium-free Tyrode's Buffer (SFTB), 2.5 μM DiSBAC₁,(3), and200 μM Direct Blue 71 (as the fluorescence quencher).

A rat pheochromocytoma (adrenal) cloned cell line, PC12, is grown inRPMI 1640 culture medium with 10% fetal calf serum (FCS), 1%penicillin/streptomycin (PS), 2 mM L-glutamine, and 1 mM sodiumpyruvate. Cells were grown in suspension, and subsequently centrifugedfrom growth medium and resuspended in DiSBAC₂(3), 1× Cell-LoadingBuffer. Approximately 100,000 cells were plated per well in a 96-wellmicrotiter plate pre-coated with poly-D-lysine to enhance cell adhesion,centrifuged at 1000 rpm for 4 minutes, and placed in an incubator for anadditional 20 minutes. Cells were not washed with any liquid medium, norwas the 1× Cell-Loading Buffer removed prior to performing fluorescencemeasurements.

The fluorescently labeled cells were analyzed for changes in membranepotential by using the FLIPR™ fluorescent imaging plate reader. Briefly,cells were depolarized with addition of 75 mM potassium gluconate insodium-containing Tyrode's Buffer (SCTB). To inhibit voltage-gatedsodium channels cells were previously incubated with 100 μM tetrodotoxin(TTX) for 5 minutes prior to depolarization. The data reveal thatcell-depolarization (due to potassium addition) causes increasedDiSBAC₁(3) fluorescence. Inhibition of sodium channels by TTX results insmaller changes in membrane potential upon potassium addition asindicated by a smaller increase in fluorescence as compared to thepositive control (75 mM potassium gluconate without TTX).

The above examples illustrate the present invention and are not intendedto limit the invention in spirit or scope.

REFERENCES

-   1. Zochowski M, Wachowiak M, Falk C X, Cohen L B, Lam Y W, Antic S,    Zecevic D., Imaging membrane potential with voltage-sensitive dyes.    Biol Bull 198, 1-21(2000).-   2. Plasek J, Sigler K, Slow fluorescent indicators of membrane    potential: a survey of different approaches to probe response    analysis. J Photochem Photobiol B 33, 101-124 (1996).-   3. Loew L M., Characterization of Potentiometric Membrane Dyes. Adv    Chem Ser 235, 151 (1994).-   4. Wu J-Y, Cohen L B., Fast Multisite Optical Measurement of    Membrane Potential. In Fluorescent and Luminescent Probes for    Biological Activity, Mason W T, Ed., pp. 389-404 (1993).-   5. Loew L M., Potentiometric Membrane Dyes. In Fluorescent and    Luminescent Probes for Biological Activity, Mason W T, 2nd Ed. Pp.    210-221 (1999).-   6. Smith J C., Potential-sensitive molecular probes in membranes of    bioenergetic relevance. Biochim Biophys Acta 1016, 1-28 (1990).-   7. Gross D, Loew L M., Fluorescent indicators of membrane potential:    microspectrofluorometry and imaging. In Methods Cell Biol 30,    193-218 (1989).-   8. Freedman J C, Novak T S. Optical measurement of membrane    potential in cells, organelles, and vesicles. Methods Enzymol 172,    102-122 (1989).-   9. Bronner C, Landry Y. The use of the potential-sensitive    fluorescent probe bisoxonol in mast cells. Biochim Biophys Acta    1070, 321-331 (1991).-   10. Shapiro H M. Cell membrane potential analysis. Methods Cell Biol    41, 121-133 (1994).-   11. Loew L M. Confocal microscopy of potentiometric fluorescent    dyes. Methods Cell Biol 38, 195-209(1993).-   12. Gonzalez J E, Tsien R Y. Improved indicators of cell membrane    potential that use fluorescence resonance energy transfer. Chem Biol    4, 269-277 (1997).

1. A test kit for measuring membrane potential changes comprising afirst fluorescent or luminescent compound having the formula

wherein X is O or S; and n is 1 or
 2. 2. The test kit of claim 1 furthercomprising a second fluorescent or luminescent reagent.
 3. The test kitof claim 2, wherein the second fluorescent or luminescent reagent iscapable of undergoing energy transfer with the first compound.
 4. Thetest kit of claim 1 further comprising a second reagent that is acalcium indicator.
 5. The test kit of claim 1, further comprising asecond reagent that is Indo-1, Fura-2, Fluo-3, Calcium Green or Fluo-4.6. A test kit according to claim 1 for measuring membrane potentialchanges further comprising a second non-fluorescent colored reagent. 7.The test kit of claim 6, wherein the second non-fluorescent reagent is afluorescence quencher.
 8. The test kit of claim, wherein n is
 1. 9. Atest kit for measuring membrane potential changes comprising a firstfluorescent or luminescent compound having the formula

wherein X is O or S; and n is 1 or 2; and a second non-fluorescentreagent that can undergo energy transfer with the first compound. 10.The test kit of claim 9, wherein the second non-fluorescent reagent isDirect Blue
 71. 11. The test kit of claim 9, wherein the secondnon-fluorescent reagent is a fluorescence quencher.
 12. The test kit ofclaim 9, wherein the second non-fluorescent reagent is not substantiallymembrane-permeant.
 13. A test kit for measuring membrane potentialchanges comprising a first fluorescent or luminescent compound havingthe formula

wherein X is O or S; and n is 1 or 2; and a second non-fluorescentreagent that can absorb the excitation light or emission from the firstcompound.
 14. The test kit of claim 13, wherein the secondnon-fluorescent reagent is not substantially membrane-permeant.
 15. Atest kit for measuring membrane potential changes comprising a firstfluorescent or luminescent compound having the formula

wherein X is O or S; and n is 1 or 2; and a second fluorescent orluminescent reagent that can undergo energy transfer with the firstcompound.